6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Water solubility

Link to relevant study record(s)

Reference

Endpoint:

water solubility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-18 to 2021-07-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Version / remarks:

adopted 24 January 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

Version / remarks:

adopted 27. Jul. 1995

Deviations:

yes

Remarks:

Consecutive fractions did not show constant concentrations within ± 30%. This was due to outliers, measured for both flow rates, and was probably caused by colloidal dissolved test item in this low concentration range.

GLP compliance:

yes (incl. QA statement)

Type of method:

column elution method

Key result

Water solubility:

ca. 1 - <= 2.22 µg/L

Conc. based on:

test mat.

Incubation duration:

> 50 - <= 70 min

Temp.:

20 °C

pH:

>= 6.1 - < 6.9

Details on results:

Refer to "Any other information on results incl. tables"

The analytical determinations are presented in the following tables:

Table 2‑a       Measurements at flow rate 25.2 mL/h

Sample	Dilution factor	Peak Area	Concentration	Difference to previous sample	pH
#		[counts*min]	[µg/L]	[%]
1	2	54240	2.16^1	-	6.31
2*	10	78044	14.59	576.3	6.34
3	2	533	2.12^1	-85.5	6.31
4	2	577	2.31^1	9.1	6.41
5	2	571	2.28^1	-1.2	6.29
6*	20	106202	45.15*	1878.4	6.23

* The values of samples 2 and 6 (grey italics) were considered as outliers and were not used in the

calculation of the mean value.

¹ Evaluated using QC recovery of 91.0 %, see Table 15.2‑g

 

Table 2‑b      Measurements at flow rate 12.4 mL/h

Sample	Dilution factor	Peak Area	Concentration	Difference to previous sample	pH
#		[counts*min]	[µg/L]	[%]
1	10	521	11.07^1	-	6.10
2	10	1460	6.57^2	-39.0	6.49
		347	6.95^1
3	2	0	<1	n.c.*	6.09
4	2	0	<1	n.c.*	6.09
5	2	0	<1	n.c.*	6.22
6	2	0	<1	n.c.*	6.17
7	2	0	<1	n.c.*	6.66
8	2	1048	4.60^3	n.c.	6.67
9	2	0	<1	n.c.*	6.88

* not calculable, because the measured value lay below the detection limit.

¹ Evaluated using QC recovery of 84.9 %, see Table 15.2‑h

² Evaluated using QC recovery of 85.9 %, see Table 15.2‑h

³ Evaluated using QC recovery of 86.8 %, see Table 15.2‑i; Sample 8 (grey italics) was considered as an outlier and was not used in the calculation of the mean value.

 

The following table gives the values which were measured for both flow rates at the plateau.

Table 3‑a       Plateau

Flow rate

Sample

Mean

Difference

[mL/h]

1

2

3

4

5

6

25.2

2.16 µg/L

14.59** µg/L

2.12 µg/L

2.31 µg/L

2.28 µg/L

45.15** µg/L

2.2 µg/L

0.09

Flow rate

Sample

Mean

Difference

[mL/h]

3

4

5

6

7

8

9

12.4

< 1 µg/L

< 1 µg/L

< 1 µg/L

< 1 µg/L

< 1 µg/L

4.60** µg/L

< 1 µg/L

<1 µg/L

n.c.*

* n.c.= not calculable, because the measured value lay below the detection limit.

** The values (grey italics) were considered as outliers and were not used in the calculation of the mean value.

Conclusions:

The water solubility of the test item was determined to be
< 1 – 2.22 µg/L at 20 ± 0.5 °C
= < 1 – 2.22 10-6 kg/m3 (SI units)
(range calculated from two flows).

Executive summary:

The solubility of the test item in water was determined by measurement of the test item concentration in the diluted test solutions using LC-MS/MS.

A pre-test to determine the water solubility with column elution was performed. Therefore, two fractions were taken from a column and measured after sample preparation. The solubility pre-test gave a water solubility of 62.26 µg/L.

Accuracy and Precision should be performed with a concentration ≤ 80 % of the value determined in the solubility test with demineralised water, which corresponds to a concentration of 49.8 µg/L. Therefore, the accuracy and precision were performed with a concentration of 40 µg/L (high level) and 4 µg/L (low level).

In the water solubility main test, the column elution method was used. Quartz sand was coated with test item and filled into an empty HPLC column with a very fine mesh from inox on both sides.

A flow rate of 25.2 mL/h was set. After discarding the first 10 column volumes, fractions were taken and measured after sample preparation. For the first 10 samples losses due to sample transfer in the measuring vials was assumed. Therefore, the following samples were collected directly into the measuring vial and measured after sample preparation.

Due to the low solubility of the test item, there are significant deviations between the measured values, but the plateau is considered as reached. The higher values were stated as outliers and were regarded to be produced by colloidal dissolved test item in this low concentration range. Nevertheless, a Tyndall effect (i.e. a laser beam was not scattered when transmitted through the liquid) could not be observed. This may also be due to the low concentration range.

The second run with a flow of approximately 12 mL/hour showed also significant deviations between the measured values, caused by outliers as in flow 1 due to the low solubility of the test item. The test was finished, because in the low concentration range obviously outliers cannot be avoided and because in several fractions of flow 2 the test item concentration lay below 1 µg/L. No Tyndall effect (i.e. a laser beam was not scattered when transmitted through the liquid) could be observed.

 

As the difference between the measured concentrations of both flow rates lay above 30%, a range is given for the solubility. No further flow rates were examined, because the concentration in flow 2 lay below the concentration of flow 1.

 

At the plateau, the concentration of the test item in water had reached:

 

< 1 – 2.22 µg/L at 20 ± 0.5 ^°C

= < 1 – 2.22 10^-6 kg/m³ (SI units)

(range calculated from two flows)

Description of key information

The water solubility was determined to be < 1 – 2.22 µg/L at 20 ± 0.5 °C. The mean value of 1.61 µg/L is used for the chemical safety assessment.

 

Key value for chemical safety assessment

Water solubility:

1.61 µg/L

at the temperature of:

20 °C

Additional information

Key

The solubility of the test item in water was determined by measurement of the test item concentration in the diluted test solutions using LC-MS/MS.

A pre-test to determine the water solubility with column elution was performed. Therefore, two fractions were taken from a column and measured after sample preparation. The solubility pre-test gave a water solubility of 62.26 µg/L.

Accuracy and Precision should be performed with a concentration ≤ 80 % of the value determined in the solubility test with demineralised water, which corresponds to a concentration of 49.8 µg/L. Therefore, the accuracy and precision were performed with a concentration of 40 µg/L (high level) and 4 µg/L (low level).

In the water solubility main test, the column elution method was used. Quartz sand was coated with test item and filled into an empty HPLC column with a very fine mesh from inox on both sides.

A flow rate of 25.2 mL/h was set. After discarding the first 10 column volumes, fractions were taken and measured after sample preparation. For the first 10 samples losses due to sample transfer in the measuring vials was assumed. Therefore, the following samples were collected directly into the measuring vial and measured after sample preparation.

Due to the low solubility of the test item, there are significant deviations between the measured values, but the plateau is considered as reached. The higher values were stated as outliers and were regarded to be produced by colloidal dissolved test item in this low concentration range. Nevertheless, a Tyndall effect (i.e. a laser beam was not scattered when transmitted through the liquid) could not be observed. This may also be due to the low concentration range.

The second run with a flow of approximately 12 mL/hour showed also significant deviations between the measured values, caused by outliers as in flow 1 due to the low solubility of the test item. The test was finished, because in the low concentration range obviously outliers cannot be avoided and because in several fractions of flow 2 the test item concentration lay below 1 µg/L. No Tyndall effect (i.e. a laser beam was not scattered when transmitted through the liquid) could be observed.

 

As the difference between the measured concentrations of both flow rates lay above 30%, a range is given for the solubility. No further flow rates were examined, because the concentration in flow 2 lay below the concentration of flow 1.

 

At the plateau, the concentration of the test item in water had reached:

< 1 – 2.22 µg/L at 20 ± 0.5 °C= < 1 – 2.22 10^-6 kg/m³ (SI units)

(range calculated from two flows)

For the purpose of the risk assessment in the CSR a mean water solubility of 1.61 µg/L was used.

 

Supporting information

In accordance with Article 25(3) of the REACH Regulation the robust study summaries submitted 12 years previously, are used as supporting information.

A water solubility of 0.021 mg/L at 20 °C (ph 6.12) was determined in a study according to EU method A.6 (Huntingdon, ECHA RSS, 2017). In a supporting study a water solubility of below 0.1 mg/L at 20 °C according to EU method A.6 was determined (Safepharm, ECHA RSS, 2017).